Measurement of IgG-blocking antibodies by ELISA using monoclonal antibody and fluorogenic substrate

An enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antihuman gamma-chain antibody and a fluorogenic substrate has been developed for quantitation of IgG-blocking antibodies in human serum. Generation of fluorescent product was linear with time to 60 min. Using optimal conditions the ELISA was sensitive to less than 1 ng/ml of specific IgG to short ragweed pollen. The assay demonstrated consistently parallel dilution curves with 51 sera (mean interdilutional coefficient of variation = 8.8%). Reproducibility was determined by constructing precision profiles for intra and interassay variation for the entire working range of the assay. Intraassay CVs ranged from a mean of 13% at threshold to less than 5% at higher antibody concentration. Interassay reproducibility similarly ranged from 18 to 10%. In this assay the effect of serum dilution on nonspecific binding was minimal and specific binding of 4-10 ng IgG antibody to the antigen-adsorbed wells was largely complete (75.8 +/- 4.8%) and highly specific (greater than 98%). This application of ELISA for ragweed IgG antibody measurement has performance specifications equal or superior to previously developed radioimmunoassay and ELISA systems.     

Virulence of Staphylococcus aureus mutants altered in type 5 capsule production.

Most clinical isolates of Staphylococcus aureus produce microcapsules (uronic acid-containing extracellular polysaccharides) that are detectable by serologic methods but are not visible by negative staining. Among the 11 reported serotypes, capsule types 5 and 8 comprise approximately 75% of all isolates. Transposon mutagenesis was performed on S. aureus to create mutants altered in capsule expression. Tn918 was introduced into the capsule type 5 strain Reynolds by filter mating, and a capsule-deficient transconjugate, JL236, was isolated. The wild-type strain was transformed with JL236 chromosomal DNA to confirm that transfer of the appropriate-size chromosomal fragment containing Tn918 generated a capsule-deficient transformant. Strain Reynolds was mutagenized with ethyl methanesulfonate to obtain a capsule-negative mutant (strain JL240). Capsular phenotypes were determined by colony immunoblots, antibody adsorption experiments, and transmission electron microscopy. The virulences of the parental and mutant strains in mice were compared. The 50% lethal doses for strains Reynolds, JL236, and JL240 were similar (10(8.59), 10(8.98), and 10(8.93) CFU, respectively). Animals injected intraperitoneally with either wild-type or mutant strains had comparable levels of bacteremia at 3 and 24 h after challenge. Quantitative cultures of blood and kidneys from animals challenged intravenously with sublethal doses of the S. aureus strains also showed no differences in bacterial clearance or renal abscess formation. These studies indicate that the type 5 S. aureus microcapsule does not promote bacterial virulence in the animal models tested.     

Changes in the level of acetylcholinesterase of Nematospiroides dubius and Trichostrongylus colubriformis following paralysis by levamisole in vivo

Acetylcholinesterase (acetylcholine acetylhydrolase; EC 3.1.1.7) levels of Nematospiroides dubius from laboratory mice and Trichostrongylus colubriformis from lambs have been measured. The anthelmintic levamisole (leavo isomer of 2,3,5,6-tetrahydro-6-phenylimidazo-(2,1b)-thiazole (Tetramizole)) did not affect the level of acetylcholinesterase in N. dubius in vivo but caused a reduction in the level of the enzyme in T. colubriformis following paralysis in vivo. The effect of levamisole on acetylcholinesterase in the nematodes is explained in terms of the differing roles of the enzyme in these two species.     

Evolutionary variants of simian virus 40: cloned substituted variants containing multiple initiation sites for DNA replication.

Two cloned evolutionary variants of simian virus 40 (SV40) containing substitutions of cellular DNA have been characterized by restriction endonuclease analysis, electron microscopic heteroduplex mapping, and DNA-DNA hybridization. Each variant genome is made up of a small, tandemly repeated segment of DNA consisting of cellular DNA and that portion of the SV40 genome containing the initiation signal for viral DNA replication. Cellular DNA sequences are different in the two variants, indicating that recombination between cell DNA and SV40-DNA can occur at more than one site. However, one end of the SV40 segment (0.68 SV40 map-units) is the same in each variant. The data suggest that substituted variants arise by integration of SV40-DNA into cellular DNA followed by excision of a small substituted genome which is subsequently amplified to a size compatible with encapsidation; the presence of multiple initiation signals in each molecule results in selective replication.     

High levels of estradiol impair spatial performance in the Morris water maze and increase 'depressive-like' behaviors in the female meadow vole

The present study investigated sex differences and the effect of a high level of estradiol in the female meadow vole on performance in the forced swim test (FST) and the Morris water maze in meadow voles. Female meadow voles were ovariectomized (OVX) and administered either vehicle (sesame oil) or estradiol for 2 days prior to performing the FST. Four days following the FST, all animals were run in the Morris water maze. Results indicated that estradiol-injected female meadow voles showed more 'depressive-like' behaviors in the FST (greater time spent immobile and less time spent swimming) than vehicle-treated female or male meadow voles. In addition, estradiol-treated females had impaired performance (greater latencies and distance swam to reach the hidden platform) than both vehicle-treated female and male meadow voles, consistent with previous data. Despite the fact that estradiol administration increased 'depressive-like' behaviors in the FST and impaired performance in the Morris water maze, there was no correlation between the two behaviors indicating that 'depressive-like' behaviors did not account for the differences seen in spatial performance in the Morris water maze. To our knowledge, this is the first demonstration in rodents indicating that estradiol-mediated changes in behavior in the FST is not indicative of subsequent performance in the Morris water maze.     

alpha-1 antitrypsin phenotypes by isoelectric focusing in a metropolitan southern Chinese population

AIMS/BACKGROUND: alpha-1 antitrypsin (alpha1AT) is an abundant protease inhibitor in human plasma. Its phenotypic variability has been reported to be associated with pulmonary emphysema and chronic liver diseases. However, alpha1AT deficiency is an uncommon condition in the Chinese population. The aim of this study was to describe the phenotypic distribution of alpha1AT in a southern Chinese population. METHODS: A total of 1085 healthy blood donors underwent alpha1AT phenotyping by isoelectric focusing. RESULTS: Two thirds (66.1%) were homozygous for either M1 or M2, whereas 32.6% were heterozygous for two different M phenotypes. The frequency of allelic variants was only 0.007, and deficiency variants were absent. Compared with earlier studies on southern Chinese populations, this study found a lower frequency of M2, and a higher number of allelic variants, including E, L, N, P, and S. This phenomenon can be attributed to population migration and mixing. CONCLUSIONS: An understanding of the alpha1AT pattern is important for evaluating the predisposition of the population to selected clinical diseases.     

Transcranial magnetic stimulation disrupts eye-hand interactions in the posterior parietal cortex

Recent neurophysiological studies have started to shed some light on the cortical areas that contribute to eye-hand coordination. In the present study we investigated the role of the posterior parietal cortex (PPC) in this process in normal, healthy subjects. This was accomplished by delivering single pulses of transcranial magnetic stimulation (TMS) over the PPC to transiently disrupt the putative contribution of this area to the processing of information related to eye-hand coordination. Subjects made open-loop pointing movements accompanied by saccades of the same required amplitude or by saccades that were substantially larger. Without TMS the hand movement amplitude was influenced by the amplitude of the corresponding saccade; hand movements accompanied by larger saccades were larger than those accompanied by smaller saccades. When TMS was applied over the left PPC just prior to the onset of the saccade, a marked reduction in the saccadic influence on manual motor output was observed. TMS delivered at earlier or later periods during the response had no effect. Taken together, these data suggest that the PPC integrates signals related to saccade amplitude with limb movement information just prior to the onset of the saccade.     

Synthesis and thermal properties of poly(enaminonitriles) containing flexible ether units

Poly(enaminonitriles) containing flexible alkyl ether units were prepared by reacting p-bis(1-chloro-2,2-dicyanovinyl)benzene with various α,ω-bis(4-aminophenoxy)alkanes in N-methyl-2-pyrrolidone (NMP). All the polymers are soluble in polar aprotic solvents such as N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N,N-dimethylacetamide (DMAc) and NMP as well as in common organic solvents such as tetrahydrofuran and acetone. High molecular weight polymers possessing inherent viscosities in the range of 0.82–1.20 dL/g were obtained and tough films were cast from a DMF solution. They showed a large exotherm around 340°C attributable to the chemical changes of the dicyanovinyl group. Thermogravimetric analyses exhibited a good thermal stability with a 10% weight loss around 450°C and 73–88% residual weight at 500°C under nitrogen.     

The high incidence of meiotic errors increases with decreased sperm count in severe male factor infertilities

BACKGROUND:The high frequency of aneuploidy sperm raises concerns that there may be an increased incidence of aneuploid offspring in ICSI programmes. In order to assess the role that chromosome complement plays in normal and abnormal fertility, detailed molecular cytogenetic studies must be done on sperm samples from men with normal and abnormal fertility. METHODS: To understand more clearly the cytogenetic make-up of sperm from oligoasthenoteratozoospermic (OAT) patients, multi-colour fluorescence in situ hybridization was used to determine numerical chromosome abnormalities. RESULTS: Increased aneuploidy frequencies for chromosomes 13, 18, 21, X and Y were detected in sperm from OAT patients. The frequencies of diploidy also increased. There were no differences in non-disjunction at meiosis I compared to meiosis II. Sperm count inversely correlated with the frequencies of diploidy, aneuploidies for chromosomes 13 and 21 in OAT patients. Twenty-two cycles of ICSI and 18 embryo transfers were performed in 20 couples. Only three cases achieved successful pregnancies. CONCLUSIONS: A higher incidence of meiotic errors and lower sperm counts was found in sperm from OAT patients.     

Characterization of knitted polymeric scaffolds for potential use in ligament tissue engineering

Different scaffolds have been designed for ligament tissue engineering. Knitted scaffolds of poly-L-lactic acid (PLLA) yarns and co-polymeric yarns of PLLA and poly(glycolic acid) (PLGA) were characterized in the current study. The knitted scaffolds were immersed in medium for 20 weeks, before mass loss, molecular weight, pH value change in medium were tested; changes in mechanical properties were evaluated at different time points. Results showed that the knitted scaffolds had 44% porosity. There was no significant pH value change during degradation, while there was obvious mass loss at initial 4 week, as well as smooth molecular weight drop of PLLA. PLGA degraded more quickly, while PLLA kept its integrity for at least 20 weeks. Young's modulus increased while tensile strength and strain at break decreased with degradation time; however, all of them could maintain the basic requirements for ACL reconstruction. It showed that the knitted polymeric structures could serve as potential scaffolds for tissue-engineered ligaments.